Phenylpropanoids including stilbenes, coumarins, and flavonoids are a major class of plant secondary metabolites that exhibit diverse molecule structures and broad pharmacological effects. Introduction of hydroxyl groups is a commonly-used strategy by nature to increase solubility, stability, structure diversity and biological activities of secondary metabolites. Recently, a variety of hydroxylated phenylpropanoid compounds were found to possess more attractive properties for potential pharmaceutical uses. For instance, piceatannol is a 3′-hydroxylated derivative of resveratrol, a well-known natural product for its anti-aging, anti-inflammatory, and cancer preventative effects (Brisdelli et al., Curr. Drug Metab., 2009, 10, 530-546). Indeed, resveratrol, as a pro-drug, is eventually converted to piceatannol in human liver by cytochrome P450 (CYP) hydroxylase (Potter et al., Br. J. Cancer, 2002, 86, 774-778), while the latter has demonstrated additional functions, e.g. tyrosine kinase inhibition, cancer cell suppression, and antiparasitic activity (Piotrowska et al., Mutat. Res., 2012, 750, 60-82). As another example, esculetin, a hydroxylated analogue of umbelliferone, has been shown to inhibit adipogenesis and induce apoptosis of maturing preadipocytes (Yang et al., Obesity (Silver Spring), 2006, 14, 1691-1699). It also inhibits tyrosinase activity and the formation of melanin (Masamoto et al., Biol. Pharm. Bull., 2004, 27, 422-425). Despite of their various functions, these hydroxylated metabolites usually exist at low abundance in nature, which hampers the exploration and application of their pharmacological properties.
Regioselective hydroxylation via synthetic chemistry approaches has been used to activate, derivatize, and functionalize inert carbons in complex compounds. However, these approaches are usually quite challenging, necessitating laborious protectiong and deprotection steps (Ullrich and Hofrichter, Cell. Mol. Life. Sci., 2007, 64, 271-293). Alternatively, biocatalytic hydroxylation provides a facile and environmental friendly way for specific oxygen transfer. In past decades, cytochrome P450 hydroxylases remained to be the dominant group of enzymes that can be engineered for this purpose (Amor eta al., Nat. Prod. Commun., 2010, 5, 1893-1898; Kille et al., Nat. Chem., 80 2011, 3, 738-743, Urlacher and Girhard, Trends Biotechnol., 2012, 30, 26-36, Urlacher and Eiben, Trends Biotechnol., 2006, 24, 324-330). However, low coupling efficiency and low activity are among the most frequently encountered problems due to the catalytic mechanism of these enzymes (Urlacher and Girhard, Trends Biotechnol., 2012, 30, 26-36; Urlacher and Eiben, Trends Biotechnol., 2006, 24, 324-330). Recently, several microbial P450 hydroxylases were identified to catalyze the orthohydroxylation of stilbenes and flavonoids (Kim et al., Drug Metab. Dispos., 2009, 37, 932-936; Lee et al., ACS Chem. Biol., 2012, 7, 1687-1692; Choi et al., Microb Cell Fact, 2012, 11, 81; Pandey et al., Enzyme Microb. Technol., 2011, 48, 386-392), however, the productivity (rate, yield and/or titer) was still low for scale-up application.